Replication of human immunodeficiency virus type 1 (HIV-1) entails a tightly regulated, complex life cycle, which is dependent upon specific interactions between viral RNAs and viral and cellular proteins. An essential and characteristic step in the viral replication cycle is the export of the intron-containing gag-pol and env mRNAs from the nucleus to the cytoplasm. The viral regulatory protein Rev mediates this event, in conjunction with the cellular nuclear export machinery and several protein cofactors. Cellular factors that are essential for Rev-directed RNA export offer alternate targets for inactivation and are potential candidates for antiviral drug development. During the past funding period, we have implicated the human Rev Interacting Protein (hRIP) and other cellular factors in a protein interaction network by which Rev mediates export of HIV RNAs from the nucleus to the cytoplasm. Our studies have revealed that hRIP is an essential cellular HIV cofactor that acts at a previously unanticipated step in Rev-directed RNA export: release of RNAs from the nuclear periphery to the cytoplasm. This perinuclear step may link nuclear export of Rev-directed RNAs with their cytoplasmic localization and function, in particular, virus assembly. Experiments are proposed to delineate the physical and functional protein interaction network mediating Rev-directed RNA export in living cells. We will use biochemical, genetic, and cell-based approaches to continue to study the function of hRIP and analyze how hRIP promotes movement of HIV RNAs from the nuclear periphery. Additionally, our studies of hRIP led us to investigate interactions between Rev and proteins that mediate cellular trafficking pathways. Experiments are proposed to explore the possibility of a functional link between Rev-directed RNA export and cellular trafficking pathways involved in HIV budding and assembly. hRIP is not required for cell viability and thus, may be an attractive target for the development of new antiviral strategies. During the past funding period, we have shown that ablation of hRIP activity using RNA interference (RNAi) results in the loss of HIV replication in mammalian cells. We will use several approaches to optimize the efficiency and specificity of RNAi-mediated silencing of hRIP expression and validate its inhibitory effect on HIV replication in mammalian cell lines and primary cells. The ultimate goal of these experiments is to validate cellular hRIP activity as a target for new antiviral strategies.